This application claims priority to German Patent Application Nos. 198 17 180.3-52 (filed Apr. 17, 1998 and issued Apr. 27, 2000 as German Patent No. DE 198 17 180) and 198 53 428.0 (filed Nov. 19, 1998), which are hereby incorporated herein by reference in their entirety.
This invention relates to a biosensor (affinity sensor) in which a hydrogel, a surfactant layer or biotin is bonded to the sensor""s precious metal surface by a short chain linker, and further relates to the procedure for the preparation thereof.
For signal generation, the surfaces of surface plasmon resonance (SPR) based affinity sensors must possess a precious metal layer, usually of gold, with a thickness of approx. 50 nm. However, direct bonding of receptors, typically proteins, to the precious metal surface has proven to be impractical because the receptors are easily denatured in this environment and as a result lose their receptor function. Also, bare sections of the precious metal surface can be subject to unspecific adsorption phenomena which seriously flaw the measurement results.
To avoid this problem, it is general practice to covalently bond to the precious metal surface a several nm thick dextran layer that swells in an aqueous medium to a thickness of approx. 100 nm and completely covers the precious metal surface. The swollen polymer layer mimics the natural environment of biomolecules and is thus suited to prevent the denaturation and the resulting inactivation of the receptors. The adsorption of molecules other than those to be analyzed is effectively suppressed. In addition, the swollen dextran layer is able to compensate surface irregularities: the bonding of receptor molecules occurs in the swollen matrix and not only immediately on the surface. This reduces the significance of surface roughness that would otherwise result in a poorly defined surface and thus in poorly quantifiable measurement results.
Coatings are known which can be applied to sensor surfaces to both prevent unspecific adsorption and to serve as a matrix for the receptor molecules (Molecular Resolution Imaging of Dextran Monolayers Immobilized on Silica by Atomic Force Microscopy, Langmuir, 1996, 12, 6436). However, such surfaces often possess defect sites resulting from their molecular structure and therefore do not provide sufficient protection against unspecific adsorption.
EP-B-589 867 describes a biosensor measuring surface in which a porous matrix, e.g. a hydrogel such as dextran, is bonded to a metal surface via a monolayer of organic molecules (linkers). Such coatings are used in the preparation of disposables for commercial SPR sensors. To ensure complete covering of the biosensor""s surface with the porous matrix and sufficient stability, EP-B-589 867 specifies that molecules having a hydrocarbon chain with a length of more than 10 atoms be used as linkers. The linker""s functional groups to which the porous matrix is bonded are hydroxyl, carboxyl, amino, aldehyde, hydrazide, carbonyl, expoxide or vinyl groups. The preferred linker is 16-mercaptohexadecanol, the hydroxyl group of which must by activated by a reaction using epichlorhydrin before the bonding of dextran to the sensor surface. EP-B-589 867 does not make any statements on the conditions of further reactions of the other functional groups with a porous matrix,
The necessity for using long-chained linkers to bond the porous matrix to the precious metal surface is a disadvantage, given the time and effort required to prepare such compounds. A further disadvantage is the use of the toxic and carcinogenic epichlorhydrin for the activation of the linker""s functional group.
Therefore, the object of the present invention is to provide a biosensor with a modified precious metal surface in which a hydrogel, a surfactant layer or biotin is linked to the precious metal surface by less complex and, if possible, commercially available linker molecules.
A further object of the invention is to provide a process for the preparation of the above biosensor with a modified precious metal surface which is less expensive and easier to implement than the state-of-the-art procedures.
These aims could be accomplished owing to the finding that even with the use of linker Molecules having a hydrocarbon chain length of 10 atoms or less, a complete covering of the precious metal surface with a hydrogel, a surfactant layer or biotin can be achieved if the linker molecule monolayer is stabilized by hydrogen bonds, aromatic-aromatic interactions or covalent bonds.
The present invention thus refers to a biosensor the surface of which comprises a precious metal layer to which a hydrogel, a surfactant layer or biotin is bonded by means of a monolayer of organic molecules, whereby said employed organic molecules used for this have the formula Axe2x80x94Rxe2x80x94B in which A is an atom or group providing the bonding to the precious metal; R is a branched or straight hydrocarbon chain with a chain length of 10 carbon atoms or less, whereby the hydrocarbon chain may be interrupted in up to two places each by a phenylene group or an heteroatom, and B is an atom or group providing the bonding to the hydrogel, the surfactant layer or the biotin.
The invention also provides a process for the preparation of the aforesaid biosensor in accordance with one of the claims 12 to 15.
The inventive biosensors with a modified precious metal surface are preferably used for surface plasmon resonance (SPR) applications.
In order to prevent unspecific adsorption of receptor molecules, the surface of the biosensor must be completely covered with hydrogel, a surfactant layer or biotin. One prerequisite for a stable hydrogel, surfactant or biotin layer is sufficient stability of the linker molecule layer underneath. Such stability can be ensured by hydrogen bonds, by aromatic-aromatic interactions (xcfx80-xcfx80 interactions) or by covalent bonds, which may be provided by an interlayer containing metal oxide. Such interactions or bonds occur between the linker molecules or between the linker molecules and the hydrogel, the surfactant or the biotin.
Hydrogen bonds may exist between amide bonds (e.g. B=amine group; carboxyl-functionalized hydrogel) or between hydroxide and carboxyl groups (e.g. B=epoxide group; carboxyl-functionalized hydrogel). In systems where hydrogen bonds exist, such hydrogen bonds may be formed between the linker modules and the hydrogel, the surfactant layer or the biotin, where they serve for bridging or preventing local defects.
Aromatic-aromatic interactions are observed between aromatic ring systems in the linker molecules. Such interactions are the source of attraction forces between the linker molecules which stabilize the monolayer. Suitable aromatic groups are phenylene groups, and in particular those phenylene groups that are incorporated in the linker molecule in para position. The phenylene groups may be substituted with small, non-bulky alkyl groups such as methyl or ethyl groups.
Another possibility to provide a stable linker molecule layer is the introduction of covalent bonds. In a preferred embodiment, the first step after bonding of a low molecular weight short-chained linker of the present invention is to build up, preferably by a sol-gel process, a dense interlayer comprising metal oxide (Stepwise Adsorption of Metal Alkoxides on Hydrolyzed Surfaces: A Surface Sol-Gel Process, Chemistry Letters, 1996, 831); which in a second step is linked to the hydrogel, the surfactant or the biotin by means of the hydroxide groups contained in it (Surface Modification for Direct Immunoprobes, Biosensors and Bioelectronics, 1996, 11, 579; Dissertation of G. Elender, Technical University of Munich, 1996, p. 113). These three references are incorporated herein by reference.
The state of the art is the stabilization of the linker molecule layer by the use of linkers with long alkyl chains that provide a dense and stable layer due to crystallization of the alkyl chains (EP-B-589 867).
One important advantage of a layered structure that makes use of hydrogen bonds or aromatic-aromatic interactions is the fact that it is very easy to prepare. On the other hand, layered structures using a metal oxide interlayer are more complex to prepare but offer the possibility of precisely controlling the layer thickness of the overall system, optionally by performing several consecutive sol-gel process steps. The layer thickness allows in turn to influence the minimum position relating to the surface plasmon resonance signal, which may be desirable from a measurement point of view.